*An Overview on* Saccharomyces cerevisiae *Indigenous Strains Selection Methods DOI: http://dx.doi.org/10.5772/intechopen.99095*

several papers in last 20 years [46, 49, 56, 69–75]. Hence, they are optimal molecular markers for the strains typing due to their size polymorphism. In general, they are useful for fingerprinting, linkage studies and knowledge on population genetic structure [5, 56, 76].

In 2016, Börlin M. et al. [74] characterised the population structure of more than 653 isolates of *S. cerevisiae* from three French cellars located at less than 10 Km from each other. Using 15 microsatellites loci as molecular markers they observed 503 different genotypes. Hence, based on SSRs analysis and using specific indexes concerning the origin of the three populations it was possible to assess a certain degree of overlapping between genotypes from two of the three cellars and the existence of a local and stable cluster of strains which shared some ancestor over 20 years. The similar composition of the *S. cerevisiae* population structure is explained by a series of events that have repeated over the years. One of these is the proximity of the wineries, which leads to a certain uniformity of the population due to the action of yeast vectors (birds, fruit flies, bees and wasps). And on the other hand, the practice of "pied de cuve", which consists in the inoculation of must with an amount of already fermenting must from a cellar to another. They noted that the SSRs-based method is more robust and sensitive compared to the inter-delta analysis, Pulsed-field Gel Electrophoresis (PFGE) and mtDNA RFPL methods [74].

Rex et al. [76] in 2020 have validated a SSRs molecular markers method for *S. cerevisiae* strain differentiation through PCR-multiplex. The method is based on two multiplex sets of primers of different size targeting polymorphic loci and it was applied on nine well characterised commercial yeasts. A set combines the six primers: ScAAT2, ScAAt3, C5, SCYOR267c, C8, C11, resulting in six different patterns after PCR and gel electrophoresis. The other one combines six other primers: YKL172w, C4, C9, ScAAT5, C6, YPL009c, resulting in five different patterns after the same process. The validation was achieved through the comparison of fragment lengths obtained by capillary sequencing and agarose gel electrophoresis image. The procedure was repeated to characterised 50 strains of *S. cerevisiae* from five different spontaneous fermentations. Through SSRs markers, 21 different new strains were recognised and characterised for their diverse aromatic profile respectively [76].

The strain identification based on SSRs polymorphisms analysis with multiplex PCR application has been used for rapid and low budget procedure too [46]. As an example, Vaudano and Garcia-Moruno [46] performed the typing of 30 commercial wine strains. The discrimination was achieved by performing a multiplex PCR using primers designed on three highly polymorphic loci: SC8132X, YOR267C and SCPTSY7 and subsequent gel electrophoresis and band pattern analysis and comparison.

Then, this analysis was employed in a dominance study between two co-inoculated strain at different temperature of fermentation, 15°C and 20°C. This trial was finalised to control the ability of these *S. cerevisiae* strains in leading the fermentation process.

Methods such as the latter can be used for applicative purpose both in oenology and in wild yeasts selection. In particular, molecular marker supports the screening of the large number of yeasts isolated from natural fermentation [75, 76].

### **2.4 Phenotype evaluation: technological characterisation, analysis of volatile compounds and sensory evaluation**

When different genotypes have been identified, the analysis of the phenotype represented by physiological tests and micro-vinification assay is the following stage of the procedure. The physiological tests are for example:

*An Overview on* Saccharomyces cerevisiae *Indigenous Strains Selection Methods DOI: http://dx.doi.org/10.5772/intechopen.99095*

production of hydrogen sulphide, killer toxin synthesis, SO2 sensitivity, nitrogen requirement [32, 77].

An interesting test consists in the *in vitro* evaluation of β-glucosidase activity. This enzyme is involved in hydrolysis of monoglucosides with the release of volatile compounds, such as benzenoid/phenylpropanoid, monoterpenes and norisoprenoides, that contribute to aromatic profile. However, β-glucosidase can affect the colour of red wine due to the lysis of anthocyanins compounds with colour alteration or loss; thus the yeast ability to modulate the anthocyanin's colour during AF must be considered in the case of red winemaking [78].

In micro-vinification, the resulting wine is then evaluated through chemical analysis of basic features and volatile compounds [45]. Then, the behaviour of the native strains selected was monitored on a pilot scale in comparison with a known yeast used as control.

An example of this pilot test has been performed in 2019 in Lebanon and aimed to identify the most efficient indigenous starter from three autochthonous *S. cerevisiae* strains previously selected during natural fermentation of Merwah wine (M.6.16, M.10.16, M.4.17). In this study, the fermentation kinetic was evaluated measuring the reduction of the density by using a hydrometer and the residual sugars were analysed by UV–visible spectrophotometry, the dominance of the strains was monitored with Inter-delta-PCR [34].

In any described cases the evaluation of technological characters (**Table 1**) at the end of AF for each indigenous strain considered was always performed, generally using official OIV methods, standards Methods (ISO) or a multiparameter analyser. The more relevant features to be considered are: fermentation trend, ethanol production (%V/V), total acidity (g/l tartaric acid equivalent), volatile acidity (g/l acetic acid equivalent), pH, free and total SO2 (mg/l), residual sugar (g/l glucose + fructose). For the microbiological stability of wine is essential a residual sugar less than 2 g/l.

Concerning the volatile acidity, it is positive a low-producer yeast, 0.2–0.4 g/l in acetic acid. High producer strains of sulphur compounds are discarded in the selection. SO2 tolerance is a positive selection criterion [79]. The killer factor is traditionally studied, but its relevance is controversial as it seems that under fermentation conditions it has no influence on sensitive yeast [80].

The evaluation of the phenotype concerns also the wine aromatic profile derived from the secondary metabolism of yeasts. The production of volatile compounds is also affected by the composition of must, in particular depending on the biochemical precursors derived from vine variety. For example, the release of the volatile thiol 4-mercapto-4-methylpentan-2-one (4MMP) from its grape-derived cysteinebound precursor is carried out by enzymes that possess carbon-sulphur lyase activity and it dependents on yeast [15].

Some volatile compounds belong to the category of higher esters and higher alcohols are shown in **Table 3** [34, 43, 48, 81–88]. In wines, esters can be formed by two different processes: fermentative ones, that involve enzymatic esterification performed by yeast, and storage for long periods that leads to chemical esterification. These two processes can concur in the synthesis of the same ester. The concentration in wine ranges from 10 to 20 mg/l. Higher alcohols are produced by yeasts, both from sugars directly and from grape amino acids through the Ehrlich reaction. They are mostly of fermentative origin and can be found in wines in quantities ranging from 150 to 550 mg/l. The main fermentative higher alcohols, part of the so-called "Fusel oils", are isobutyl alcohol (2-methyl-propan-1-ol) and amyl alcohols (mixture of 2-methyl-butan-1-ol and 3-methyl-butan-1-ol). At concentration lower than 300 mg/l they participate in the aromatic complexity of the wine; at higher concentrations their penetrating odour masks the wine's aromatic finesse.


*An Overview on* Saccharomyces cerevisiae *Indigenous Strains Selection Methods DOI: http://dx.doi.org/10.5772/intechopen.99095*


#### **Table 3.**

*Some volatile compounds from* S. cerevisiae *metabolism, respective odour descriptors, olfactory threshold and common concentration in wine.*

#### **Figure 5.**

*Comparison of sensory profiles of two (A and B) red wines fermented with two different indigenous strains of*  S. cerevisiae*.*

Acetic esters of these alcohols, especially isoamyl acetate, have a banana fragrance that may play a positive role in the aroma of some young red wines (primeur or nouveau) [79].

Usually, the analysis of volatile is performed by gas chromatography equipped with Mass Spectrometer as detector (GC–MS) [43, 48, 81–88].

The last examination at the end of a pilot scale production is the sensory evaluation performed by a panel test. That consist in the personal evaluation of wine

descriptors fulfilled by a group of judges trained in the recognition of organoleptic features (appearance, odour, taste, texture) (ISO 1993). The panel, in short, quantifies the level of descriptors using an intensity scale as required by the ISO 2003 standard b. The sensory session must be performed in standard condition of the room, glasses, temperature, time, so that the environment does not affect the judges [34, 43, 48, 81–88]. An example of sensory analysis results is shown in **Figure 5**. This sensory examination could be useful to predict the consumer appreciation. At the end of this process, all the data obtained by every test must be statistically analysed. The strain or strains which show the best performance and which better meet the enologist's preferences, can be used in an industrial scale assay.
